Question

(LC) Describe how to prepare a LC ODS column. (includes the support, stationary phase synthesis and packing). How to vary the column polarity of supported silica? Why sometimes it needs end-caping?

Answer 1

Liquid Chromatography, or LC, is performed using a solid support stationary phase (also called a column).   ODS(octadecyl-silica or C18) is the most popular methods of silica-based stationary phases used in reverse phase chromatography.

The goal of creating a column is to resolve two or more compounds in space based on the difference in polarity of the compounds, and the column must be of uniform diameter, density, and length.

Support/ base: microparticulate silica support

Stationary phase：octadecyl-silica or C18 coated on top of the support

Particle ：2 µm, 3 µm, 5 µm Pore ：120 Å [general specifications]

The mobile phase is a liquid that moves through the packed bed of stationary phase in the column under pressure.

Step 1: Packing a column

1. Glass wool: To prevent the solid material from flowing through the stopcock when opened.

2. Sand : Provides level surface to build a silica column on.

3. Solvent: To prevent the disturbance of sand

4. Slurry: which also contains the stationary phase.

mobile phase silica gel sand plug

The mobile phase is now drained out from the top due when the stopcock from the bottom is opened and the mobile phase exits the column through the porosities of the stationary phase. Then, the mixture of compounds is inserted from the top while the stationary phase(gray) is not dry and then some more mobile phase is inserted on top of it.

The compounds of the two mixtures separate in the stationary phase on the basis of the difference in the polarities. The less polar compound drops more and vice versa.

Varying the column polarity of supported silica

This can be achieved by altering the interactions between the particles of the stationary phase, for eg, through hydrogen-bonding and electron donor-acceptor interactions and making changes in the particle size.

End-capping

Hydroxyl groups are originally attached to the surface of the silica gel carrier in a Si-OH structure. This is called "silanol." ODS packing is formed by bonding ODS groups to the silanol through chemical reactions. However, as the ODS group is bulky and not highly reactive, a lot of unreacted silanol remains.

As this residual silanol can have a variety of adverse effects during analysis, the silanol is capped by reactions with trimethylsilyl (TMS) groups, which are not bulky but are highly reactive. This operation is known as end-capping.